The subject matter disclosed herein relates to acquisition of microscopy images, such as of biological or histological samples. More particularly, the disclosed subject matter relates to the calibration and focus of microscopes used in such image acquisition protocols.
Certain types of molecular pathology examinations utilize a multiplexing workflow for molecular pathology imaging. When generating images using such a multiplexing workflow, a single slice of tissue (i.e., a single sample) may be used. The multiplexing workflow allows images of the tissue sample acquired over multiple rounds of imaging to be layered, with each round of imaging being directed to a different set of biomarkers applied to the sample. Through the combination of biomarkers acquired over multiple rounds of imaging, a comprehensive view of tissue composition may be attained for the sample.
By way of example, in one such approach the tissue sample is repeatedly stained and bleached during a given imaging protocol. A round of image acquisition may be performed using an automated fluorescence microscope for background acquisition or after each stain or bleach cycle. For example, after an initial stain application a set of images may be acquired, after which the sample may be bleached and stained with the next biomarker and another set of images acquired.
With this in mind, one problem arising from microscopy imaging in general, including multiplexed approaches such as the one described, is how to calibrate (e.g., optically, geometrically, mechanically, and so forth) certain aspects of an image acquisition by a microscope. For example, if a tissue slide is not loaded properly into a slide holder, there is a risk of tilted slide loading and possible out-of-focus images during an automated image acquisition. In particular, when a slide is loaded into a slide holder tilted and the tilt has exceeded the focusing range of the objective for imaging, all or part of some images may be out of focus.
Prior attempts are characterizing the planar tilt of a slide have depended on utilizing image-based focus at a number of points to determine a base focus height, and then calculate a tilt from this. However, this approach only works for locations on the slide where tissue exists, and is therefore an incomplete solution. Approaches are needed that are more versatile, that are more accurate or as accurate, and which are both faster and less computationally intensive.